POTASSIUM CHANNEL BLOCKING COMPOUNDS AND THEIR USE

Abstract: Specific potent transient outward potassium channel inhibitors, and polypeptides derived from spider venom, and uses thereof.

Description: DESCRIPTION POTASSIUM CHANNEL BLOCKING COMPOUNDS AND THEIR USE Field of the Invention This invention relates to inhibitors of the transient outward potassium channel and other potassium channels. Background of the Invention The following is a description of relevant art, none of which is admitted to be prior art to the claims- Potassium (K + ) channels are membrane-spanning proteins that allow the selective movement of K + into or out of cells in response to changes in membrane potential, or in response to activation by cations and/or ligands. The primary role of K + channels is maintenance of the resting membrane potential; another role concerns their contribution to repolarization of action potentials in excitable cells. Potassium channels represent a diverse group of ion channel proteins, and several toxins have been described that act primarily (if not exclusively) by blocking one or more specific K + channels (Rudy, "Diversity and Ubiquity of K Channels", 25 Neuroscience 729, 1988) . Cardiac cells are characterized by a remarkable variety of different K + channel subtypes. Several K + channels types are opened in response to depolarization of the membrane during an action potential, and the currents carried by these different channels sum to cause repolarization of the membrane to the resting potential. One of these chan- nel types, the transient outward K + channel, conducts a current (I 10 ) which activates very rapidly (within 1-10 milliseconds) upon membrane depolarization and then decays (inactivates) rapidly (10-200 milliseconds) . I l contri¬ butes most significantly to initial repolarization of the cardiac action potential, and is blocked by several non- specific pharmacological compounds such as aminopyridines and tedisamil, a class III antiarrhythmic agent (Dukes et al., "Tedisamil Blocks the Transient and Delayed Rectifier K + Currents in Mammalian Cardiac and Glial Cells", 254 J. Pharmacol. Exp. Ther. 560, 1990) . Blockage of I l0 leads to prolongation of the cardiac action potential. I l0 has been recorded from human cardiac cells as described, for example, in Escande et al., "Two Types of Transient Outward Currents in Adult Human Atrial Cells", 252 Am. J. Physiol. H142, 1987. Lengthening of cardiac action poten- tials (and hence refractoriness) is said to be one mecha¬ nism for suppression of reentrant atrial and ventricular arrhythmias (Lynch et al., "Therapeutic Potential of Modulating Potassium Currents in the Diseased Myocardium", 6 FASEB J. 2952, 1992) . Currently available class III antiarrhythmic agents, most of which act by blocking a separate subtype of K + channels known as delayed rectifier K + channels, may cause excessive prolongation of cardiac action potentials that can lead to the development of a ventricular arrhythmia, known as torsades de pointes (Sanguinetti, "Modulation of Potassium Channels by Antiar¬ rhythmic and Antihypertensive Drugs", 19 Hypertension 228, 1992) . The opening of voltage-dependent K + channels is also the mechanism by which repolarization of the cell membrane occurs during the very short action potential characteristic of central neurons. Transient outward K + currents (referred to as I Λ in neurons) play a role in this process. Dendrotoxin, a toxin derived from snakes, selectively blocks the delayed non-inactivating K + current in dorsal root ganglion neurons (Penner et al., "Dendrotoxin: A Selective Blocker of a Non-inactivating Potassium Current in Guinea-pig Dorsal Root Ganglion Neurones", 407 Pflugers Arch. 365, 1986) , and also blocks the transient outward K + current (I A ) in hippocampal slices (Halliwell et al., "Central Action of Dendrotoxin: Selective Reduction of a Transient K Conductance in Hippocampus and Binding to Localized Acceptors", 83 Proc. Natl. Acad. Sci. USA 493, 1986) . Prolongation of action potential duration in neurons by dendrotoxin results in an enhanced release of neurotrans itters (Harvey and Anderson, "Dendrotoxins: Snake Toxins That Block Potassium Channels and Facilitate Neurotrans itter Release", 31 Pharmac. Ther. 33, 1985) . It has been suggested that fur¬ ther work in this field may confirm this as a pharmaco¬ logical approach to the treatment of cognitive disorders such as Alzheimer's disease (Lavretsky and Jarvik, "A Group of Potassium-channel Blockers- Acetylcholine Releasers: New Potentials for Alzheimer Disease? A Review", 12 J. Clinical Psychopharm. 110, 1992). Summary of the Invention This invention generally concerns specific and potent novel inhibitors or blocking agents of the tran¬ sient outward potassium channel, for example, currents referred to as I lo or I Λ in cardiac cells and in neurons, respectively. The invention also features novel poly¬ peptides isolated from spider venom, or their equivalent, which are active at one or more potassium channels, e.g., the transient outward potassium channel. Specifically, examples are provided of the novel activity of polypeptide toxins isolated from the venom of the spiders Heteropoda venatoria , and Olios fasciculatus These peptides, referred to simply herein as Compounds 1, 2 and 3 are examples of specific and potent blockers of voltage-dependent transient outward K + channels, which block the corresponding whole-cell current (I lo ) of cardiac cells. As such, these agents, fragments thβreof, or compounds discovered using a ligand binding assay (or its equivalent) employing these toxins, or their equivalent, are useful in the treatment of cardiac arrhythmias and have utility in the treatment of disorders of learning and memory, (such as Alzheimer's disease) , Parkinson's disease, multiple sclerosis, schizophrenia, epilepsy, stroke and muscle spasticity. In general, useful K + channel inhibiting poly¬ peptides of the type described and claimed herein can be isolated from the venom of the spiders Heteropoda venatoria and Olios fasciculatus . Other polypeptides (or their equivalent) with similar or homologous amino acid (or other compound or monomer) sequences that block potassium channels can also be isolated. Applicant believes it is the first to determine the existence of such K + channel blocking activity in toxins derived from spider venom, and thus demonstrates that it is useful and productive to screen for other such polypeptides in spider venom. This invention also concerns methods of using these polypeptides to screen for other agents acting at a common site (i.e. , the transient outward potassium channel) as active agents. Chemical agents that selectively block I Λ channels in central neurons, and thereby cause enhanced release of neurotransmitters, are believed by applicant to be useful in the treatment of Alzheimer's disease and other neural disorders. Similarly, agents which selec- tively block I lo channels in cardiac cells are believed by applicant to be useful in treatment of cardiac arrhyth¬ mias. Thus, these polypeptides and related compounds or agents can be used for the treatment of cardiac arrhyth¬ mias, Alzheimer's disease, Parkinson's disease, multiple sclerosis, schizophrenia, epilepsy, stroke and muscle spasticity. Thus, in a first aspect, the invention features specific potent transient outward potassium channel inhi¬ bitors, blockers or antagonists. , The phrase "transient outward potassium channel" is a well recognized phrase which defines a specific sub¬ type of potassium channels within a variety of cells, e.g. , as characterized by the currents I, u and I Λ noted above, in cardiac and neural cells, respectively. The term "inhibitor" is used to mean agents which reduce current conducted by transient outward potassium channels. In the art, terms such as "blocker" and "antagonist" have been used interchangeably with the term "inhibitor". By "specific" is meant that the blockage of transient outward K + channels is half-maximal (ICj o )at or below 100 nM, and that no effect on other K + channels (such as delayed rectifier, inward rectifier, acetylcholine- activated or ATP-inhibited K + channels) , Na + or Ca 2+ channels occurs at concentrations at least 10-fold greater than the ICj,, for transient outward K * channels. By "potent" is meant that a given transient out¬ ward K + channel is blocked by 50% at a concentration of an inhibitor less than 100 nM, more preferably less than 10 nM, and even more preferably less than 1 nM. In preferred embodiments, these agents are poly- peptides or are derived from polypeptides present in spider venom. While specific examples of such polypep¬ tides are provided herein, these examples are not limiting in this invention and those of ordinary skill in the art will recognize that other polypeptides can be readily iden- tified within various spider venoms, including but not limited to those described herein. In addition, those of ordinary skill in the art will recognize that equivalent polypeptides can be synthetically formed using standard procedures. Specific portions of those peptides which are active in blocking, inhibiting, or antagonizing a selected transient outward potassium channel can be readily iden¬ tified using standard screening procedures. For example, specific peptide fragments can be synthesized, or produced from intact polypeptides using various peptidases, and those fragments assayed for inhibitory activity in assays, as described below. Those fragments which are active as inhibitors are useful in this invention in various screen¬ ing assays, and in therapeutic applications. In addition, analogues or uteins of such poly¬ peptides can be readily synthesized. These may contain modifications in the amino acid sequence in regions which do not affect the inhibitory or blocking activity of the original polypeptide. In more conserved regions of the inhibitor, amino acids may be substituted such that the activity of the inhibitor is not significantly altered, for example, by substitution of small amino acids, such as glycine, for other small amino acids, such as valine, or positively or negatively charged amino acids for similarly charged amino acids. The term "derived" simply indicates that com- pounds can be synthesized based upon the general structure of polypeptides identified in spider venom. Such deriva- tization is performed by methods well recognized in the art, specific examples of which are generally provided above. Preferably, the term "derived" includes analogues, muteins and fragments, as described above, which have a desired modulatory activity of a polypeptide toxin des¬ cribed herein. The above inventions are exemplified by poly¬ peptides found in the venom of the spiders Heteropoda venatoria and Olios fasciculatus . Three specific poly¬ peptides of this invention and the fractions in which they are present according to this invention include Heteropoda venatoria peptide Compound 1 (SEQ. ID. NO. 1) , Heteropoda venatoria peptide Compound 2 (SEQ. ID. NO. 2) , and Olios fasciculatus peptide Compound 3 (SEQ. ID NO. 3) . In one example, polypeptides of this invention block transient outward K + channels in cardiac and neural cells. This invention includes polypeptides which have substantially the same amino acid sequence, and substantially the same K + current blocking activity as the polypeptides Heteropoda venatoria peptides Compound 1 and Compound 2, and Olios fasciculatus peptide Compound 3. In a second aspect, the invention features a method for screening for a transient outward potassium channel active agent by contacting a transient outward potassium channel with a known specific transient outward potassium channel inhibitor (such as those described above) and a potential transient outward potassium channel active agent, and detecting inhibition of binding of the known inhibitor by the potential active agent. Inhibition of binding is an indication of a useful transient outward potassium channel active agent. Such active agents can be readily screened to determine their specificity. An "active agent" is a compound that either increases (if it acts as an agonist) or decreases (if it acts as an inhibitor) current through a transient outward potassium channel. In a related aspect, the invention features a method for screening spider venom for a useful K + channel active agent as exemplified by methods described herein. Such venom is screened to determine fractions which con- tain the desired activity. By "K + channel active agent" is meant a compound that either increases or inhibits any other type of K + channel such as delayed rectifier, inward rectifier, Ca 2" "- activated or ATP-sensitive K + channels. In preferred embodiments, the screening method involves use of transient outward potassium channels from cardiac or neural tissue. Also within the scope of this invention is a method for identifying compounds that bind to the tran- sient outward K + channel, preferably at the same site as that bound by the Heteropoda venatoria peptides Compound 1 and Compound 2, and Olios fasciculatus peptide Compound 3. In a third aspect, the invention features a method for treatment of a disease or condition in which a therapeutically useful result is achieved by modulating a transient outward potassium channel activity, by ad inis- tering to the organism a therapeutically effective amount of a specific transient outward potassium channel inhibi¬ tor, or a polypeptide (or its equivalent) from spider venom. Specific diseases to be treated include (but are not limited to) those listed above. By "modulating" is meant a decrease of transient K + channel activity. For example, by blocking the pore of the channel, or by changing the voltage dependance of channel gating. Treatment involves the steps of first iden¬ tifying a patient (human or non-human) that suffers from a disease or condition by standard clinical methodology and then providing such a patient with a therapeutically available composition of the present invention. By "therapeutically effective" is meant an amount that relieves (to some extent) one or more symptoms of the disease or condition in the patient. Additionally, by "therapeutically effective" is meant an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with or causative of the disease or condition. Generally, it is an amount between about 1 nmole/kg and 1 μmole/kg of the molecule, dependent on its EC, υ and on the age, size, and disease associated with the patient. In a further aspect, the invention features a pharmaceutically acceptable composition including a specific transient outward potassium channel inhibitor or spider venom polypeptide (or its equivalent) . In yet a further aspect, the invention features a polypeptide (or analogues thereof) obtainable from a spider venom, that is an inhibitor of potassium channel activity. The invention also features unique fragments of such a polypeptide. Such analogues, as defined above, are not themselves obtained from the venom but are derived by analysis of an isolated polypeptide (as exemplified herein) or can be obtained by screening other venoms. The term "unique fragments" refers to portions that find no identical counterpart in known sequences as of the date of filing this application. These fragments can be identified easily by an analysis of polypeptide data bases existing as of the date of filing to detect counterparts. _. By "pharmaceutically acceptable composition" is meant a therapeutically effective amount of a compound of the present invention in a pharmaceutically acceptable carrier, i.e., a formulation to which the compound can be added to dissolve or otherwise facilitate administration of the compound. Examples of pharmaceutically acceptable carriers include water, saline, and physiologically buf¬ fered saline. Such a pharmaceutical composition is pro- vided in a suitable dose. Such compositions are generally those which are approved for use in treatment of a speci¬ fied disorder by the FDA or its equivalent in non-U.S. countries. By "disease or condition" is meant those diseases listed above and related diseases concerning cardiac or neural cells. Also within the scope of the invention is the use of such compounds as insecticidal agents. The compounds described herein are believed by Applicant to possess significant insecticidal action, perhaps through block of a channel that is structurally similar to the transient outward K + channel of mammalian heart muscle. Spiders are known to produce venoms that contain a variety of toxins with potent insecticidal activities (Quistad, G.B., et al. "Insecticidal activity of spider (Araneae) , centipede (Chilopoda) , scorpion (Scorpionidae) , and snake (Serpentes) venoms", 85 Journal Economic Entomology 33, 1992) . These toxins have evolved in response to evolutionary pressures to produce effective toxins that effectively and rapidly kill or paralyse prey insects (Jackson, H. and P.N.R. Usherwood, "Spider toxins as tools for dissecting elements of excitary amino acid transmission", 11 Trends in Neurosciences 278, 1988) . Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiments The drawings will first briefly be described. Drawings Fig. 1 is a chromatograph of Heteropoda venatoria venom (120 μl) fractioned on a Vydac C18 reverεed-phase HPLC column (10 x 250 mm) equilibrated in 80% A/20% B.. Elution with a linear gradient from 76% A/24% B to 65% A/35% B over 44 min. (A=0.1% TFA (aq) , B=0.1% TFA in CH--CN) . The gradient was begun 5 minutes after injection of the venom and interrupted at 39 min to take the column to 50% B over 3 minutes to elute the final peaks. The absorbance of the eluant was monitored at 220 nm. The fractions (#1 - 8, and the end fraction) are identified by number at the bottom of the absorbance traces. Fig. 2 is a chromatogram of peptide Compounds 1 and 2 fractionated on a cation-exchange column. (Panel a) For peptide Compound 1 the column was developed with a linear gradient from 0-0.32 M NaCl in 50 mM sodium acetate, pH 4.0 in 32 min followed by a linear gradient from 0.32-1 M NaCl in 50 mM sodium acetate, pH 4.0 in 5 min. (Panel b) For peptide Compound 2 the column was developed with a linear gradient from 0-0.3 M NaCl in 50 mM sodium acetate, pH 4.0 in 3 min followed by a linear gradient from 0.3-1 M NaCl in 50 mM sodium acetate, pH 4.0 in 35 min. Elution was at 1 ml/min and the effluent was monitored at 280 nm. Fractions were collected as noted on the chromatogram. Fig. 3 is a chromatogram of Olios fasciculatus venom (108 μl) fractionated on a Vydac C-18 reversed phase column (300 A, 10 x 250 mm) . Five minutes after injection of the sample, the column was developed with a linear gradient from 20-45% acetonitrile/0.1% TFA in 75 minutes. At 50 minutes, the column was taken to 100% acetonitrile/0.1% TFA over 7 min. The flow rate was 3.0 ml/min and the effluent was monitored at 220 nm. Fractions were collected as noted on the chromatogram. Fig. 4 is a chromatogram of peptide Compound 3 purified by cation-exchan • ■• - chromatography. Five minutes after injection of the sample, the column was developed with a linear gradient from 0.25-1 M NaCl in 50 mM sodium acetate buffer, pH 4.0 in 75 min. Elution was at 1 ml/minute and the effluent was monitored at 280 nm. Fractions were collected as noted on the chromatogram. The following is a detailed description of the methods and tests by which useful compounds of this inven- tion can be discovered and utilized for treatment of, e.g. , cardiac arrhythmias and disorders of memory and learning. A key method is the means by which compounds, both synthetic and natural products, can be rapidly screened with radioligand binding techniques (or their equivalent) to identify those which modify binding at the site on the transient outward K + channel bound by Compound 1, Compound 2, or Compound 3. Screening for K + channel blockers from spider venom can be performed in a similar manner. In addition, useful derivatives or analogues or muteins of such polypeptides can be readily screened using this methodology. Additional testing will include whole-cell re¬ cording of ionic currents in cardiac and neural cells to confirm that agents which compete with radiolabeled Com- pound 1, Compound 2, or Compound 3 binding act as specific active agents of transient outward K + currents, and are without significant effect on other channel types. The desired properties of agents identified by means outlined in this invention include: 1) Specific and potent block of a potassium channel, e.g. , cardiac or neural transient outward K + channels. Specific block implies that the agent will not have demonstrable effects on other ionic channels or receptors at concentrations in vitro that block I l0 or I A at dosages that prove to be of therapeutic utility in the treatment of cardiac arrhythmias or disorders of learning and memory, or the other diseases listed above. 2) Lack of significant side effects at thera¬ peutic doses, including excessive prolongation of QT interval of the electrocardiogram, bradycardia, hyper- excitability or seizures. Isolation of Spider Venom Toxins The following is a non-limiting example of methods by which inhibitory spider toxins of this inven¬ tion may be isolated. Those in the art will recognize that equivalent methods can be used to isolate and iden- tify other polypeptides (or their equivalent) having useful activity in this invention. Equivalent compounds are those referred to herein as analogues, muteins and derivatives which are identified by methods described herein as having useful activity of one or more K + channels. Those in the art will recognize that once one useful K + channel active agent is identified and sequenced it can be chemically synthesized in totality or as frag¬ ments, and modifications in the sequence made, as described below. In addition, such fragments or the poly- peptide itself may be used to screen for other such active agents which are equivalent in their activity to the original polypeptide. Venom is obtained from the spiders Heteropoda venatoria and Olios fasciculatus through the process of milking by electrical stimulation according to standard methods well known to those skilled in the art. It is preferred that the method employed is one which safeguards against contamination of the whole venom by abdominal regurgitant or hemoly ph. Such methods are wel| known to those skilled in the art. The whole venom so obtained is stored in a frozen state at about -78°C until used for purification as described below. Purification of the constituents from the whole venom is accomplished by reversed-phase high performance liquid chromatography (HPLC) on a variety of preparative and semi-preparative columns such as C-4 and C-18 Vydac columns (Rainin Instrument Co. Inc., Mack Road, Woburn, Massachusetts 01801) . Peak detection is carried out monochromatically at 220 nm. Further analysis of the fractions can be accomplished with, for example, polychrome UV data col¬ lected with a Waters 990 diode array detector (Millipore Corporation, Waters Chromatography Division, 34 Maple Street, Milford, Massachusetts 01757) . The fractions from the columns are collected by known methods such as through the use of a fraction collector and an ISCO 2159 peak detector (ISCO, 4700 Superior, Lincoln, Nebraska, 68504) . The fractions are collected in appropriately sized vessels such as sterile polyethylene laboratory ware. Concentra¬ tion of the fractions is then accomplished by lyophiliza- tion from the eluant followed by lyophilization from water. Purity of the resulting constituent fractions then can be determined by chromatographic analysis using a different type of column than the system used in the final purification of the fractions. Peptide Sequencing The polypeptides of the invention, e.g. , identi- fied as described herein, can be sequenced according to known methods. A general strategy for determining the primary structure includes, for example, the following steps: 1) Reduction and S-pyridylation of disulfide- bridged cysteine residues to enhance substrate suscepti- bility to enzymatic attack; 2) Controlled cleavage of the peptide through single or multi-step enzymatic digestion; 3) Isolation and purification of peptide fragments via reversed-phase high performance liquid chromatography (HPLC) ; 4) Characterization of peptide fragments through N-terminal sequencing and ion-spray mass spectrometry. S-pyridylethylation of cysteine residues of the polypeptides under study can be performed, for example, in solution followed by amino acid sequencing of the polypep¬ tides. One such procedure for S-pyridylethylation can be accomplished as described below. About 1 to 10 μg of polypeptide is dissolved or diluted in up to 50 μl of a buffer prepared by mixing 1 part TrisHCl, pH 8.5, containing 4 mM EDTA and 3 parts 8M guanidineHCl. 2.5 μl of 10% aqueous 2-mercaptoethanol is added and the mixture is incubated at room temperature in the dark under argon for two hours. After incubation, 2 μl of 4-vinylpyridine (fresh reagent stored under argon at - 20°C) is added and the mixture is incubated for another two hours at room temperature in the dark under argon. The mixture is then desalted, preferably by chro¬ matography on a short reversed-phase column. The re- covered alkylated polypeptide is then sequenced according to known methods. Alternatively, the polypeptide can be sequenced after in si tu reduction and S-pyridylethylation as described in Kruft et al., 193 Anal. Biochem. 306 (1991) . Given the benefits of the disclosure herein with respect to the peptides Compound 1 and Compound 2 from the venom of Heteropoda venatoria , and peptide Compound 3 of Olios fasciculatus it is now possible to obtain other peptides by methods other than through isolation/purifica- tion from whole venom. The polypeptides of this invention can be produced using reco binant DNA techniques through the cloning of a coding sequence for said polypeptides or portions thereof. For example, hybridization probes which take advantage of the now known amino acid sequence infor- mation of said polypeptides can be employed according to methods well known to those skilled in the art to clone a coding sequence for the entire polypeptide. A combination of recombinant DNA techniques and in vitro protein syn¬ thesis can also be employed to produce the polypeptides of this invention. Such in vitro protein synthesis methods include, but are not limited to, use of an ABI 430A solid phase peptide synthesizer (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, California 94404) employing standard Merrifield chemistry or other solid phase chemistries well known to those skilled in the art. Equivalent Peptides It is well known in the art that certain amino acid substitutions can be made in polypeptides which do not affect, or do not substantially affect, the function of said polypeptides. The exact substitutions which are possible vary from polypeptide to polypeptide. Determina- tion of permissible substitutions is accomplished accord¬ ing to procedures well known to those skilled in the art. Thus, all polypeptides having substantially the same amino acid sequence and substantially the same K + channel blocking activity are within the scope of this invention. Biological Activity The polypeptides or fragments thereof are useful in the treatment of cardiac arrhythmias or in disorders of memory and learning such as Alzheimer's disease and those other diseases noted above. When used for such indica- tions, the peptides and fragments are formulated according to standard formulation methods known in the art, such as those disclosed in Remington's Pharmaceutical Sciences (latest edition, Mack Publishing Company, Easton, PA) . The nature of the formulation will depend on the route of administration and the dosage required. Optimization of the dosage for a particular indication can be accomplished using standard optimization techniques as is generally practiced for peptide medicaments. In general, administration by injection is pre- ferred, either intravenous, intramuscular, subcutaneous or intraperitoneal. For injection, the peptide or fragments are formulated in liquid medium, such as Ringer's solu¬ tion, Hank's solution, or other forms of physiological saline. Formulations may also involve lyophilized prepar- ations which can be reconstituted for administration. Alternative means of providing the active compounds of the invention to the subject include transmucosal and trans- dermal administration, wherein the formulation includes a permeation enhancer, such as a detergent, as well as addi¬ tional excipients. Properly formulated, oral administra¬ tion is also within the scope of the invention. Screening Assay Employing Peptides Compounds 1, 2 and 3 In addition, the peptides and biologically active fragments thereof are useful in screening assays to assess the ability of small molecules or other candidate drugs to inhibit the binding of Compounds 1, 2 or 3 to car¬ diac or neural transient outward K + channels. Described herein below is a suitable assay for competitive binding in which the Compound 1, 2 or 3 of the invention are useful. For use in this assay, generally, the polypep¬ tides Compound 1, 2 or 3 (or an active fragment) is sup- plied in radiolabeled form and the ability of the candidate compound to compete with radiolabeled Compound 1, 2 or 3 is assessed. The following examples are intended to illus¬ trate but not to limit the invention. Example 1: Heteropoda venatoria Venom Fractions Approximately 900 μl of Heteropoda venatoria venom was fractionated by diluting 75-120 μl aliquots of the crude venom to 1 ml with A and applying the diluted fractions to a Vydac C18 column (10 x 250 mm) equilibrated in 20% B. (A=0.1% TFA (aq) ; B=0.1% TFA in CH--CN) . After 3 min, the gradient was changed to 24% B over 1 min and at 5 min, a linear gradient from 24% to 35% B over 44 min was begun. The flow rate was 3.5 ml/min and the effluent was detected at 220 nm (Fig. 1) . The following fractions were collected: fraction 1 (peaks eluting between 5 and 16 minutes) , fraction 2 (peaks eluting between 16 and 19 minutes) fraction 3 (peaks eluting between 19 and 23.5 minutes) fraction 4 (peaks eluting between 23.5 and 26.5 minutes) fraction 5 (peaks eluting between 26.5 and 29.5 minutes) fraction 6 (peaks eluting between 29.5 and 33 minutes) fraction 7 (peaks eluting between 33 and 37 minutes) fraction 8 (peaks eluting between 37 and 39 minutes) , and the end fraction (peaks eluting between 39 and 46 minutes) . At 39 min, after the majority of the venom components had eluted, the column was taken to 50% B over 3 min. When no further peaks eluted (-7 minutes) the column was returned to 20% B over 4 min and equilibrated for the next chromatography. Like fractions from the 8 chromatographic runs were combined and lyophilized. The major peaks of fractions 6 and 7 correspond to compounds 1 and 2, respectively described in Examples 2 and 4 below. Example 2: Heteropoda Peptide Compound 1 Crude Heteropoda venatoria venom (-50 μl) was applied to a reversed phase HPLC column (Vydac, C-18 300 A, 22 x 250 n ) and was operated using a biphasic linear gradient program from 80% A and 20% B to 65% A and 35% B over 60 minutes (A = 0.1% trifluoroacetic acid (TFA) , B = acetonitrile) with detection at 220 nm and a flow rate of 15 ml/min. The desired fraction was collected from 43 to 44 min. Pooled fractions from individual runs were con¬ centrated by lyophilization. The structure of peptide Compound 1 was deter¬ mined and verified by the following methods. PTC amino acid analysis was carried out on 1-10 nmols in triplicate using the Waters Pico-Tag system. N-terminal sequencing was carried out on a pulse-liquid sequenator (ABI) on both native and reduced/pyridylethylated peptide. Mass spectral analysis was obtained from a SCI-EX API III ion spray mass spectrometer. A pyridylethylated derivative of Compound 1 suitable for N-terminal sequencing was generated in situ according to the method of Kruft et al., 193 Anal. Biochem. 306 (1991). The data taken together affirm the structure of peptide Compound 1 as shown below. SEQ. ID. NO. 1: Asp Asp Cys Gly Lyε Leu Phe Ser Gly Cys Asp Thr Asn Ala 1 5 10 Asp Cys Cys Glu Gly Tyr Val Cys Arg Leu Trp Cys Lys Leu 15 20 25 Asp Trp 30 30 residues, 6 cysteines, 3 disulfide bonds, Calculated mass = 3412.86 (amide) . Observed mass = 3412.70 (ion spray m.s.) . Estimated pi = 3.76. Example 3: Heteropoda Peptide Compound 1 Peptide Compound 1 was also further purified by cation-exchange chromatography on a HEMA-IEC BIO SB column (10 μm, 4.6 x 150 cm; Alltech Associates, Deerfield, IL 60015) . The lyophilized material containing peptide Compound 1 from the reversed-phase chromatography was dissolved in three ml of 50 mM sodium acetate, pH 4.0 and chromatographed in three equal portions as follows. One ml was loaded onto the HEMA-IEC BIO SB column equilibrated in 50 mM sodium acetate, pH 4.0. After 5 min, the column was developed with a linear gradient from 0-0.32 M NaCl in 50 mM sodium acetate, pH 4.0 in 32 min followed by a linear gradient from 0.32-1 M NaCl in 50 mM sodium acetate, pH 4.0 in 5 min (Fig. 2a) . After 10 min, the column was returned to the starting conditions in 5 min and equilibrated for subsequent chromatographies. Elution was at 1 ml/min and the effluent was monitored at 280 nm. Fractions were collected as noted on the chromatogram. The remaining two ml of crude Compound 1 was chromatographed as described above and like fractions from the three chromatographies were combined. The major absorbance peak, which eluted from the cation-exchange column between 26.5 and 29 min, was desalted on a Vydac C-18 reversed-phase column (10 x 250 mm, 300 A) . The pooled fraction (-10 ml) was loaded onto the reversed-phase column equilibrated in 20% acetonitrile/0.1% TFA. After 10 min, the column was developed with a linear gradient from 20-35% acetonitrile/0.1% TFA in 30 min at a flow rate of 3.5 ml/min and the effluent was monitored at 220 nm. The fraction eluting between 35.5 and 38 min was lyophilized to give 641 μg of purified peptide Compound 1. The observed mass of this peptide was 3412.72 (electrospray ionization) . Example 4 : Heteropoda Peptide Compound 2 Crude Heteropoda venatoria venom (-50 μl) was applied to a reversed-phase HPLC column (Vydac, C-18, 300 A, 2? > :250 mm) and was operated using a biphasic linear gradient program from 80% A and 20% B to 65% A and 35% B over 60 min (A = 0.1% trifluoroacetic acid (TFA), B = acetonitrile) with detection at 220 nm and a flow rate of 15 ml/min. The desired fraction was collected from 46 to 48.5 min. Pooled fractions from individual runs were concentrated by lyophilization. The material from the fractionation above, derived from 50 μl of crude venom, was applied to a reversed-phase HPLC column (Vydac, C-18, 300 A, 22 x 250 mm) and was operated using an isocratic program of 75% A and 25% B + (A = 0.1% TFA, B = acetonitrile) with detec¬ tion at 220 nm and a flow rate of 3.5 ml/min. The desired fraction was collected from 55 to 68 min. Pooled like fractions from individual runs were concentrated by lyophilization. The structure of peptide Compound 2 was deter¬ mined and verified by the following methods. PTC amino acid analysis was carried out on 1-10 nmols in triplicate using the Waters Pico-Tag system. N-terminal sequencing was carried out on a pulse-liquid sequenator (AB,I) on both native and reduced/pyridylethylated peptide. Mass spec¬ tral analysis was obtained from a SCI-EX API III ion spray mass spectrometer. A pyridylethylated derivative of Compound 2 suitable for N-terminal sequencing was generated in situ according to the method of Kruft et al., 193 Anal. Biochem. 306 (1991). The data taken together affirm the structure of peptide Compound 2 as shown below. SEQ. ID. NO. 2: Glu Cys Gly Thr Leu Phe Ser Gly Cys Ser Thr His Ala Asp 1 5 10 Cys Cys Glu Gly Phe lie Cys Lys Leu Trp Cys Arg Tyr Glu 15 20 25 Arg Thr Trp 30 31 residues, 6 cysteines, 3 disulfide bonds. Calculated mass = 3599.05 (amide) . Observed mass = 3599.38 (ion spray m.s.) Estimated pi = 5.41. Example 5: Heteropoda Peptide Compound 2 Peptide Compound 2 was also purified by cation- exchange chromatography on a HEMA-IEC BIO SB column (10 μm, 4.6 x 150 cm) . The lyophilized material containing peptide Compound 2 from the initial reversed-phase chromatography was dissolved in three ml of 50 mM sodium acetate, pH 4.0 and chromatographed in three equal portions as follows. One ml was loaded onto the HEMA-IEC BIO SB column equilibrated in 50 mM sodium acetate, pH 4.0. After 5 min, the column was developed with a linear gradient from 0-0.3 M NaCl in 50 mM sodium acetate, pH 4.0 in 3 min followed by a linear gradient from 0.3-1 M NaCl in 50 mM sodium acetate, pH 4.0 in 35 min (Fig. 2b) . After 5 min the column was returned to the starting conditions over 10 min and equilibrated for subsequent chromatographies. Elution was at 1 ml/min and the effluent was monitored at 280 nm. Fractions were collected as noted on the chromatogram. The remaining two ml of crude Compound 2 was chromatographed as described above and like fractions from the three chromatographies were combined. The major absorbance peak, which eluted from the cation-exchange column between 30 and 34 min, was desalted in two portions on a Vydac C-18 reversed-phase column (10 x 250 mm, 300 A) . The column was equilibrated in 25% acetonitrile/0.1% TFA and eluted with the starting solvent for 10 min, followed by a linear gradient from 25-35% acetonitrile/0.1% TFA in 20 min at a flow rate of 3.5 ml/min. The effluent was monitored at 220 nm and peptide Compound 2 eluted as a single peak from 26.5 to 29 min. The remaining pool from the cation-exchange column was then desalted and like fractions were combined. This pool was lyophilized to give 1.88 mg of purified peptide Compound 2. The observed mass of this peptide was 3599.52 (electrospray ionization) . Example 6: Fractionation of Olios fasciculatus venom Approximately 108 μl of Olios fasciculatus venom was fractionated by diluting the whole venom with 1.5 ml of 20% acetonitrile/0.1% TFA and loading the sample on to a Vydac C-18 column (300 A, 10 X 250 mm) equilibrated in the same buffer. Five minutes after injection of the sample, the column was developed with a linear gradient from 20-45% acetonitrile/0.1% TFA in 75 min (Fig. 3) . At 50 min, after the majority of the venom components had eluted, the column was taken to 100% acetonitrile/0.1% TFA over 7 min. The flow rate was 3.0 ml/min and the effluent was monitored at 220 nm. Fractions were collected as noted on the chromatogram. The fraction (#21) containing peptide Compound 3, which eluted between 40 and 42 min, was lyophilized and the residue dissolved in 2 ml of 50 mM sodium acetate, 0.25 M NaCl, pH 4.0. Example 7 : Olios fasciculatus Compound 3 Peptide Compound 3 was further purified by cation exchange chromatography on a HEMA-IEC BIO SB column (10 μ , 4.6 x 150 cm, from Alltech Associates, Deerfield, IL 60015) . The solution containing peptide Compound 3 (1.5 ml) from the reversed-phase chromatography was loaded onto the HEMA-IEC BIO SB column equilibrated in the same buffer. After 5 minutes, the column was developed with a linear gradient from 0.25-1 M NaCl in 50 mM sodium acetate buffer, pH 4.0 in 75 min (Fig 4). Elution was at 1 ml/min and the effluent was monitored at 280 nm. Fractions were collected as noted on the chromatogram. The major peak of material (fraction #4) , which eluted from the cation-exchange column between 27 and 32 min, was desalted on a Vydac C-18 reversed-phase column (10 x 250 mm, 300 A) . The fraction (-4.5 ml) was loaded onto the reversed-phase column equilibrated in 0.1%TFA. After 3 min, the column was developed with a linear gradient from 0-15% iso-propanol/0.1% TFA in 3 min followed by a linear gradient from 15-30% iso- propanol/0.1% TFA in 30 min and from 30-50% iεo- propanol/0.1% TFA in 5 min. Elution was at 1.0 ml/min and the effluent was monitored at 220 nm. The fraction eluting between 43 and 45 min was lyophilized to give 70 μg of purified peptide Compound 3. The observed mass of this peptide was 3786.64 (electrospray ionization) . N-terminal sequence analysis was obtained for reduced, derivatized peptide Compound 3. The sequence is as follows: SEQ. ID. NO. 3: Asp Asp Cys Ala Gly Trp Met Glu Ser Cys Ser Ser Lyε 1 5 10 Pro Cys Cys Ala Gly Arg Lys Cys Phe Ser Glu Trp Tyr 15 20 25 Cys Lys Leu Val Val Asp Gin Asn 30 34 residues, 6 cysteineε, 3 disulfide bonds. Observed mass = 3786.64 (ion spray m.s.) There is low confidence in the identity of amino acids 33 and 34. Example 8: K + Channel Blocking Activity of Heteropoda venatoria and Olios fasciculatus Peptide Fractions, and Compounds 1, 2 and 3 The ability of the peptide fractions and Compoundε 1, 2 and 3 of this invention to block transient outward K + channels is demonstrated by the following procedure. Rat ventricular myocytes were isolated according to the procedure described previouεly (Kamp et al., "Voltage- and Uεe-dependent Modulation of Cardiac Calcium Channels by the Dihydropyridine (+)-202-791" , 64 Circ. Res. 338, 1989) . The method involves retrograde perfusion of an excised rat heart with a solution containing collagenase and protease to enzymatically digest the entire heart so as to isolate individual cardiac myocytes suitable for use in standard voltage clamp experiments. Whole-cell currents are recorded from isolated myocytes using the voltage clamp techniques described in detail elsewhere (Hamill et al., "Improved Patch Clamp Techniques for High-resolution Current Recording from Cells and Cell-free Membrane Patches", 391 Pflugers Arch. 85, 1981) . Cells are placed in a 0.5 ml recording chamber and bathed in a buffered solution of the following composition (in mM) : NaCl, 132; MgCl 2 , 1.2; CaCl 2 , 1.8; KC1, 4; HEPES, 10; glucoεe, 10; pH = 7.4. In most experiments in which K" currents were recorded, Ca 2+ current was blocked by omission of CaCl 2 and addition of 1 mM Co 2+ to this solution. The myocytes are voltage clamped using a commercially available patch clamp amplifier (Axon Instruments Axopatch ID) , and data acquisition and analysiε is performed using a personal computer. Cellε were clamped at a potential of -60 V. Teεt potentials (500 msec duration) were applied to potentials ranging from -40 to +30 mV. Using these techniques several K * currents can be recorded in these cells, including an inward rectifier K + current; a rapidly activating, non-inactivating delayed rectifier K + current; and a voltage-dependent transient outward K + current (I l0 ) . The dried fraction residues of venom fractions 1-8 and the end fraction prepared as described in Example 1 were each dissolved in 1 ml of water. A 10 μl sample of each was then diluted with 3 ml of the buffered solution to test for effects on cardiac K + currents. Under these con- ditions, peptide fractions 2-9 blocked I , in a voltage- dependent manner. Block was complete at a test potential of -10 V, with block reduced to 30 to 70% of control values at a test potential of +30 mV. The predominant peptides of fractions 6 (Compound 1) and 7 (Compound 2) were isolated and purified as described in Examples 3 and 5 above. Compound 1 blocked I, n in a voltage-dependent manner, with greater block occuring at lesε depolarized test potentials. This was quantified by determining the concentration required to inhibit I 10 by 50% (IC M ) and the maximum block of this current at three different test potentials. The maximum block of I,„ at test potentials of -10 mV, +20 mV and +50 mV was 100%, 79% and 69%, respectively. The IC*. for block of I lo was 16 nM at -10 mV, 35 nM at +20 V, and 138 nM at +50 mV (n = 4-6) . Consistent with block of I, 0 , Compound 1 (30 nM) prolonged action potential duration, measured at 90% repolarization, of isolated rat ventricular myocytes by 34±5% (n=5) . Selective prolongation of action potential duration represents class III antiarrhythmic activity (Vaughan Williams, "Delayed ventricular repolarization as an antiarrhythmic principle", 6 Eur. Heart J. 145, 1985) . The effects of Compound 1 or 2 on other cardiac currents was determined to asseεε their εpecificity. At concentrations of 0.2 - 1.0 μM, Compound 1 or Compound 2 did not affect the following cardiac currents as meaεured uεing εtandard whole cell-voltage clamp techniques: -ultrarapid delayed rectifier K 1 current (I Ku .) in rat ventricular myocytes -slow delayed rectifier K + current (I Ks ) in guinea pig ventricular myocytes -inward rectfier K^ current (I κι ) in rat and guinea pig ventricular myocytes -sodium current (I N in rat ventricular myocytes -L-type Ca + current (I C .. L ) i n rat and guinea pig ventricular myocytes In an isolated human ventricular myocyte, Compound 1 (0.2 μM) also blocked I lo but not I Kur , εimilar to the findings in rat ventricular myocytes. Compound 3 had similar activity, blocking I to completely at test potentials < 0 mV at 1 μM, while having no effect on either delayed rectifier or inward rectifier K + currents. The effects of Compound 1 and Compound 2 (1 μM) were substantially reversed upon washout of the toxins. Compound 1 or 2 was also tested for activity on a number of other K + channels recorded from isolated non- cardiac cells. At 0.2 - 1.0 μM, Compound 1 or 2 had no effect on: -rapid delayed rectifier K + currents (I κ ) of rat neural cellε (Purkinje neurons, cerebellar granule cells, hippocampal pyramidal cells, sympathetic ganglion cells) , GH 3 pituitary cells, or rabbit osteoclastε. -transient outward current (I Λ ) of rat cerebellar granule cells or sympathetic ganglion cells. -a cloned channel (Kvl.4) expressed in Xenopus oocytes. Thus, Compounds 1 and 2 were shown to be quite specific for one type of channel (a voltage-activated, transient outward K' current) in cardiac myocytes. The only other toxin reported to inhibit a transient outward K 4 current (in neural cellε) iε dendrotoxin (Haliwell et al., "Central action of dendrotoxin: Selective reduction of a transient K conductance in hippocampus and binding to localized acceptors", 83 Proc. Natl. Acad. Sci. USA 493, 1986) . However, we have shown that dendrotoxin (2 μM) has no effect on rat cardiac I l0 . Therefore, Compounds 1, 2 and 3 are the first toxins described that specifically block cardiac I l0 . Example 9: Neural effects of Compound 1 and 2 The ability of Compounds 1 and 2 of this invention to affect neural activity is demonstrated by their electrophysiological effects on hippocampal slices. Male Sprague-Dawley rats (100-200 g) were sacrificed by decapitation. The brain was removed from the cranium, immediately placed in cold (4-6°C) , oxygenated (95% 0 2 /5% C0 2 ) artificial cerebrospinal fluid (aCSF) conεiεting of (in mM) NaCl, 126; KC1, 2.5; NaH 2 P0 4 , 1.24; MgS0 4 , 1.3; CaCl 2 , 2.4; NaHCO,, 26; glucoεe, 11, and εlices of hippocampus were prepared as described previously (Mueller et al., "Arylamine spider toxins antagonize NMDA receptor-mediated synaptic transmisεion in rat hippocampal εlices", 9 Synapse. 244, 1991) . Slices were maintained in a reservoir of 200 ml of oxygenated aCSF at room temperature. Following a 1 hr recovery period, a single εlice waε tranεferred to a εmall volume (-300 μl) recording chamber. Small platinum weightε were placed on the εlice to increaεe the εtability of the recording. The εlice was covered with aCSF and a superfusion system maintained the flow of fresh, oxygenated aCSF at 2 ml/min. The slice was held submerged in the flow of aCSF so that potential problems with accesε of drugε into the slice were minimized. The temperature in the recording chamber was held at 33°C for extracellular field potential recording. Bipolar concentric stimulating electrodes were placed under visual guidance in the stratum radiatum near the border of CA1-CA2. To evoke synaptic reεponses, monophaεic 50 μsec pulseε of 3-50 V were delivered to the εlice every 30 εec while teεting the reεponεe until potentialε of maximal amplitude were obtained from a particular recording εite. The voltage waε then εet εo aε to evoke a half-maximal response. Recording was done with 2-3 MW glass microelectrodes filled with 0.9% NaCl, which were also placed under visual guidance. Synaptic responεeε were recorded from the CA1 pyramidal cell layer (population εpike) or from stratum radiatum (field excitatory postsynaptic potential (EPSP) and afferent volley (AV) ) , digitized, and entered into an IBM PC-based data acquisition and storage syεtem. Toxins were made up in phosphate-buffered saline (PBS: NaCl, 140 mM; KCl, 2.5 mM; KH 2 P0 4 , 1.5 mM; Na 2 HP0 4 , 8.1 mM; pH 7.4) at 100-1000 times the desired final concentration, and then trans¬ ferred to the reservoir syringe so as to achieve, via dilution and mixing, the desired final concentration. All drugs and toxins were applied by superfusion for 30 min, at which time the response amplitude had generally plateaued. All waveforms were digitized and stored on disk. Extracellularly recorded response amplitudes were averaged over a 5 min period just prior to drug application (control, pre-drug) and again over a 5 min period following drug application (25-30 min poεt-drug) . Compound 1 produced a εustained increase in the population spike amplitude which did not recover during washout with fresh aCSF. The mean responεe to 500 nM Compound 1 was a 35 ± 9% increase (mean ± S.E.M. , n = 5 sliceε) . In two of these sliceε, the amplitude of the εimultaneouεly recorded field EPSP waε increased by an average of 16%, while the afferent volley was unchanged. Similar resultε were obtained uεing purified Compound 2, which produced an increaεe in the population εpike amplitude of 25 ± 7% (mean + S.E.M. , n = 9 εliceε) when applied at a final concentration of 1 μM. In two of theεe εliceε, the amplitude of the εimultaneouεly recorded field EPSP waε increaεed by an average of 12%, while the afferent volley waε unchanged. Taken together, these results demonstrate that Compounds 1 and 2 produce long-term increases in synaptic transmisεion at the Schaffer collateral-CAl pyramidal cell εynapεe. These data cannot distinguish between a pre- and post-synaptic site of action, nor can they point clearly to a mechanism of action. Such increases in synaptic transmisεion could be due to blockade of voltage-εenεitive potaεεium channels. Many K 4 channel blocking agents can cause seizureε when injected intravenously (i.v.) , or when administered by intracerebroventricular (i.e.v.) injection. For example, dendrotoxin causeε convulεionε and death in mice when injected i.e.v. at 0.008 μg/g, equivalent to about 0.24 μg/mouεe (Schweitz, H. et al., "Purification and pharmacological characterization of peptide toxinε from the black mamba (Dendroaεpis polylepiε) venom", 28 Toxicon 847, 1990) . In contraεt, Compound 2 did not cauεe convulεions or seizureε in audiogenic εeizure-prone mice injected i.e.v. with 1 μg (n=3) or 2 μg (n=l) of Compound 2. After i.v. injection at doεeε of 10, 15 or 24 μg (n=l each doεe) , Compound 2 cauεed a tranεient ataxia in mice, but convulεions were not observed. Example 10: Other K + Channel Blocking Toxins Compounds 1, 2 and 3 repreεent the firεt reported exampleε of toxinε isolated from spider venoms that block specific K + channels. Venoms from species of spiders other than Heteropoda venatoria and Olios fasciculatus may contain structurally unrelated toxins (peptides and nonpeptides) that potently block I [0 or other typeε of K + channelε in mammalian cellε. Several other toxinε isolated from venoms of invertebrate and vertebrate venoms have been well characterized. For example, two toxins have been isolated from venom of the bee Apis mellif ra that block K + channelε. Apamin blockε a low conductance Ca 2+ 7 activated K + channel, whereaε MCD (maεt cell degranulating) peptide blockε a non-inactivating delayed rectifier K + channel (Strong, "Potassium Channel Toxins", 46 Pharmac. Ther. 137, 1990) . Other K + channel specific blocking toxins have been isolated from venoms produced by scorpions and snakes. For example, venom from the scorpion Leiurus quinquestriatus contains at least two toxins, charybdo- toxin and leiurotoxin that block high conductance, and low conductance Ca 2+ -activated K + channels, respectively (Strong, "Potassium Channel Toxins", 46 Pharm. Ther. 137, 1990) . Toxins that block non-inactivating delayed rectifier K + channels of neurons have also been isolated from the venoms of mamba snakes (Harvey and Anderson, "Dendrotoxins: Snake Toxins That Block Potassium Channels and Facilitate Neurotransmitter Release", 31 Pharmac. Ther. 33, 1985) . Dendrotoxin from the green mamba snake (Dendroaspis angusticepε) and Toxin 1 from the black mamba (D . polylepis) both share considerable sequence homology with -bungarotoxin, an inhibitory presynaptic neurotoxin isolated from venom of Bungaruε multicinctuε that alεo blocks the same type of K + channel (Moczydlowski et al., "An Emerging Pharmacology of Peptide Toxinε Targeted Againεt Potaεεiu Channelε", 105 J. Membrane Biol. 95, 1988) . The above noted toxinε are reported to have effects beyond block of non- inactivating delayed rectifier K + channels. For example, /3-bungarotoxin also exhibits phospholipase A2 activity (Moczydlowεki et al.) and dendrotoxin also blocks sodium current and a slow inactivating transient K + current in hippocampal neurons (Li and McArdle, "Dendrotoxin Inhibits Sodium and Transient Potassium Currents in Murine Hippocampal Neurons", 64 Biophvs. J. A198, 1993) . The above-noted toxins have been useful in defining the role of specific K + channels in the physiology of normal cells and cells of diseased tissues. However, there are several K + channels known for which no highly specific and potent modulators have been discovered. Spider venoms repreεent an untapped εource for the discovery of such novel channel ligands. The existence of K + channel-specific toxins in spider venoms is examined by testing the effects of whole venoms, venom fractions separated by standard HPLC methodology, and isolated toxins on K + currents measured uεing standard whole-cell voltage clamp recording techniques on iεolated mammalian cardiac and neural cellε aε described in Example 8 above. Example 11: Method for Screening Compoundε that Bind to Compound 1/Compound 2/Compound 3 Site on the Transient Outward K + Channel in Neural Tissue Compound 1,2 or 3 or related peptides are labeled with 12S I by procedures known in the art (lactoperoxidase, Bolton-Hunter, chloramine T, etc.) . Candidate compoundε acting at the Compound 1/Compound 2/ Compound 3 binding site are asεeεεed by determining their ability to displace specific binding of [ 125 I]Compound 1, [ 12S I]Compound 2, or ['^I]Compound 3 or related peptides labeled with l2 I using techniques described below. The following assay can be utilized as a high throughput asεay to screen product libraries (e.g. , natural product libraries and compound files at major pharmaceutical companies) to identify new classes of compounds with activity at the Compound 1/Compound 2 /Compound 3 binding site on the I, 0 channel. These new classes of compounds are then utilized as chemical lead structures for a drug development program targeting the Compound 1/Compound 2/Compound 3 binding site on the neural I l0 channel. The compounds identified by this assay offer a novel therapeutic approach to disorderε of learning and memory such as Alzheimer's diseaεe, and thoεe other diseases listed above. It is important to demonstrate that a peptide retainε itε biologica.1 activity if it iε to be uεed in a quantitative binding assay. Iodinated ( l27 I) Compound 1, Compound 2, and Compound 3 retain their normal activity with regard to block of cardiac I 1O . For example, ι:7 I-Compound 1 blocked I u , of rat ventricular myocytes in a voltage-dependent manner, with approximate IC^'s of 25 nM at -10 mV, 70 nM at +20 mV, and 150 nM at +50 mV. Rat brain membraneε are prepared according to the method of Williamε et al. ("Effects of Polyamines on the Binding of [ 3 H]MK-801 to the NMDA Receptor: Pharmacological Evidence for the Exiεtence of a Polyamine Recognition Site", 36 Molec. Pharmacol. 575, 1989) as followε: Male Sprague-Dawley ratε (Simonεen Laboratorieε) weighing 100-200 g are sacrificed by decapitation. The brains from 20 rats (minus cerebellum and brainste ) are homogenized at 4°C with a glaεε/Teflon homogenizer in 300 ml 0.32 M εucroεe containing 5 mM K-EDTA (pH 7.0) . The homogenate is centrifuged for 10 minutes at 1,000 x g and the supernatant removed and centrifuged at 30,000 x g for 30 minutes. The resulting pellet is resuεpended in 250 ml 5 mM K-EDTA (pH 7.0) εtirred on ice for 15 minuteε, and then centrifuged at 30,000 x g for 30 minutes. The pellet is resuspended in 90 ml 5 mM K-EDTA (pH 7.0) , and 15-ml aliquots are layered over diεcontinuouε εucroεe gradientε of 0.9 M and 1.2 M εucroεe (10 ml each) . The gradients are centrifuged at 95,000 x g for 90 minutes, and the synaptic plasma membrane (SPM) fraction at the 0.9 M/1.2 M sucrose interface collected. Membranes are washed by resuεpenεion in 500 ml 5 M K-EDTA (pH 7.0) , incubated at 32°C for 30 minutes, and centrifuged at 100,000 x g for 30 minutes. The wash procedure, including the 30 minutes incubation, is repeated three times. The final pellet is resuspended in 60 ml 5 mM K-EDTA (pH 7.0) and stored in aliquots at -80 ° C. To perform a binding asεay with [ 12S I]Compound 1, 2 or 3, aliquotε of εynaptic plaεma membraneε (SPMε) are thawed, washed once by incubation at 32 ° C for 30 minutes, and centrifuged at 100,000 x g for 30 minutes. SPMs are resuspended in buffer A (20 mM K-HEPES, 1 mM K-EDTA, pH 7.0) . The [ 125 I]Compound 1, 2 or 3 is added to this reaction mixture. Binding assayε are carried out in polypropylene test tubeε. The final incubation volume iε 200 μl. Nonspecific binding is determined in the presence of 100 μM nonradioactive Compound 1, 2 or 3. Triplicate sampleε are incubated at 32°C for 2 hours. Assays are terminated by the addition of 10 ml of ice-cold buffer A, followed by filtration over glasε-fiber filterε (Schleicher & Schuell No. 30) . The filters are washed with another 10 ml of buffer A, and radioactivity iε determined by gamma counting for 125 I. In order to validate the above assay, the following experiments are also performed: (a) The amount of nonspecific binding of the [ l25 I]Compound 1, 2 or 3 to the filters is determined by passing 200 μl of buffer A containing 100 nM [ 125 I]Compound 1, 2 or 3 through the glasε-fiber filters. The filters are washed with another 10 ml of buffer A, and radioactivity bound to the filters iε determined by εcintillation counting. If a εignificant amount of nonεpecific binding of the [ 125 I]Compound 1,2 or 3 occurε, then filterε are prewaεhed with unlabeled Compound 1, 2 or 3 to limit thiε binding. If high nonεpecific binding remainε a problem, aεεays will be terminated by centrifugation rather than by filtration, and the amount of radioactivity in the pellet will be determined by scintillation counting. (b) A saturation curve is constructed by resuεpending SPMε in buffer A. The assay buffer (200 μl) contains 75 μg of protein. Nine concentrations of [ 12S I]Compound 1, 2 or 3 are used, ranging from 10 nM to 100 μM in half-log units. A εaturation curve iε conεtructed from the data, and an apparent K**, value and B maλ value determined by Scatchard analyεiε (Scatchard, "The Attractionε of Proteinε for Small Moleculeε and Ions", 51 Ann. N.Y. Acad. Sci. 660, 1949) . The cooperativity of binding of the [ l25 I]Compound 1, 2 or 3 is determined by the conεtruction of a Hill plot (Hill, "A New Mathematical Treatment of Changes of Ionic Concentrations in Muscle and Nerve Under the Action of Electric Currents, With a Theory to Their Mode of Excitation", 40 J. Physiol. 190, 1910) . (c) The dependence of binding on protein (receptor) concentration is determined by reεuεpending SPMε in buffer A. The aεsay buffer (200 μl) contains a concentration of [ 125 I]Compound 1, 2 or 3 equal to its K D value and increasing concentrations of protein. The specific binding of [ 15 I]Compound 1, 2 or 3 should be linearly related to the amount of protein (receptor) present. (d) The time course of ligand-receptor binding is determined by resuspending SPMs in buffer A. The assay buffer (300 μl) contains a concentration of [ I25 I]Compound 1, 2 or 3 equal to its K„ value and 100 μg of protein. Triplicate samples are incubated at 32°C for varying lengths of time; the time at which equilibrium is reached is determined, and this time point is routinely used in all subsequent assays. (e) The pharmacology of the binding site can be analyzed by competition experiments. In such experiments, the concentration of [ l2<i I]Compound 1, 2 or 3 and the amount of protein are kept constant, while the concentration of teεt (competing) drug iε varied. Thiε assay allows for the determination of an IC S0 and an apparent K D for the competing drug (Cheng and Prusoff, "Relationεhip Between the Inhibition Conεtant (K-) and the Concentration of Inhibitor Which Causes 50 Percent Inhibition (IC Sϋ ) of an Enzymatic Reaction", 22 J. Biochem. Pharmacol. 3099, 1973) . The cooperativity of binding of the competing drug is determined by Hill plot analysis. Specific binding of the [ 12S I]Compound 1, 2 or 3 represents binding to a novel site on the I 1O channel. As such, peptides related to Compound 1, 2 or 3 εhould compete with the binding of [ l2S I]Compound 1, 2 -or 3 in a competitive faεhion, and their potencies in this assay should correlate with their inhibitory potencies in a functional assay of I l0 block (e.g. , inhibition of I, in isolated neural or cardiac cells) . Conversely, compoundε which have activity at the other εiteε on the I l0 channel should not displace [ I25 I]Compound l, 2 or 3 binding in a competitive manner. Rather, complex allosteric modulation of [ 125 I]Compound 1, 2 or 3 binding, indicative of non- competitive interactionε, might be expected to occur. (f) Studieε to eεti ate the dissociation kinetics are performed by measuring the binding of [ 125 I]Compound 1, 2 or 3 after it is allowed to come to equilibrium (see (d) above) , and a large excesε of nonradioactive competing drug iε added to the reaction mixture. Binding of the [ l2S I]Compound 1, 2 or 3 iε then assayed at various time intervals. With this assay, the asεociation and disεociation rateε of binding of the [ I25 I]Compound 1, 2 or 3 are determined (Titeler, "Mul tiple Dopamine Receptors : Receptor Binding Studieε in Dopamine Pharmacology", Marcel Dekker, Inc., New York, 1983) . Additional experiments involve varying the reaction temperature (20°C to 37°C) in order to understand the temperature dependence of these parameters. Example 12 : Method for Screening Compounds that Bind to Compound 1/Compound 2/Compound 3 Site on the Transient Outward K "1" Channel in Cardiac Tissue Compound 1, Compound 2, Compound 3 and related peptides are labeled with I2 I by procedures known in the art (lactoperoxidase, Bolton-Hunter, chloramine T, etc.) . Candidate compoundε acting at the Compound 1/Compound 2/ Compound 3 binding εite are assessed by determining their ability to displace specific binding of [ 12" 'I]Compound 1, [ 12 I]Compound 2, [ ll, I]Compound 3 or related peptides labeled with 125 I using techniques described below. The following asεay can be utilized ιaε a high throughput aεsay to screen product libraries (e.g. , natural product libraries and compound files at major pharmaceutical companies) to identify new clasεes of compounds with activity at the Compound 1/Compound 2/ Compound 3 binding site on the cardiac I t0 channel. These new classes of compounds, are then utilized as chemical lead εtructures for a drug development program targeting the Compound 1/Compound 2 binding/Compound 3 site on the cardiac I lo channel. The compounds identified by thiε aεεay offer a novel therapeutic approach to the treatment of reentrant εupraventricular and ventricular cardiac arrhythmiaε. Cardiac εarcole mal veεicleε are prepared according to the method of Doyle et al. ("Saxitoxin binding and "Faεt" Sodium Channel Inhibition in Sheep Heart Plasma Membrane", 249 Am. J. Physiol. H328, 1985) and Joneε and Beεch ("Iεolation of Canine Cardiac Sarcolemmal Veεicleε", 5 Methodε in Pharmacology 1, 1984), aε modified by Kamp and Miller ("Voltage-dependent Nitrendipine Binding to Cardiac Sarcolemmal Vesicles", 32 Mol. Pharmacol. 278, 1987) . Cardiac sarcolemmal vesicleε are prepared from fresh bovine or other suitable mammalian heart tisεue at 0-4°C. The heart iε cut into 1 cm 3 pieceε, and converted into a paεte with a meat grinder. The paεte was homogenized in 4-times its volume of 0.75 M choline Cl buffered to pH 7.4 with 30 M IV-2-hydroxyethylpipera- zine-W-2- ethanesulfonic acid (HEPES) -15 mM Tris. The homogenization was carried out twice for 30 seconds in 300 ml polypropylene centrifuge jars with a Tekmar T185 shaft. Thiε and all other bufferε include the proteinase inhibi¬ tors: 0.2 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, and 1 mM dithiothreitol. The reεultant homogenate is centrifuged for 20 minuteε at 27,000 x g in the GSA rotor of a Sorvall centrifuge. The εupernatant iε diεcarded, and the pellet iε reεuεpended in 10 mM HEPES-5 mM Triε, pH 7.4 and recentrifuged as before. The pellet from this centrifugation is resuspended in 10 mM HEPES,-Tris and homogenized three times for 30 εecondε each with the T185 εhaft of the Tekmar at a εetting of 5. The resulting homogenate is centrifuged for 20 minutes in the GSA rotor at 14,000 x g. The εupernatant is then centrifuged in the GSA rotor at 27,500 x g for 70 inuteε. After the preliminary centrifugationε the membraneε are suspended in 50% sucroεe, 150 mM KCl, 100 mM TriεCl, and 5 mM Na pyrophosphate. These vesicleε are loaded onto the bottom of a four εtep discontinuous gradient with additional steps at 30%, 21.5%, and 9.5% sucroεe. Thiε gradient iε centrifuged at 1.5 hour at 193,000 x g in a Beck an 50.2Ti rotor. The pellicle at the 9.5%-21.5% εucroεe interface iε enriched in εurface εarcolemma. Thiε pellicle iε collected and diluted into a buffer containing 150 mM KCl, 0.8 mM MgS0 4 and 10 mM TrisCl (pH=7.4 @ 22°C), then centrifuged for 35 minutes at 193,000 x g. The resulting pellet is resuεpended in loading buffer and centrifuged once again. The final pellet is resuεpended in loading buffer to a final protein concentration of 2 mg protein/ml, frozen in liquid N 2 and εtored at -70°C until uεe. Membrane veεicleε (20-40 μg protein) are loaded with 150 mM KCl and diluted 50-fold into 1 ml of binding buffer containing 150 mM KCl. The veεicleε are preincubated 5 minutes at 37°C and then incubated for an additional time required for the attainment of equilibration conditions (exact time determined by preliminary experiments) in the presence of varying concentrations of [ l2S I]Compound 1, 2 or 3 (1 nM-1 μM) . The binding reaction iε terminated by addition of 4 ml of ice cold binding buffer and then rapid filtration over Whatman GF/C filterε followed by three additional 4 ml waεheε with ice cold binding buffer. The radioactivity aεεociated with the filterε iε determined using standard gamma counting techniques. Specific [ ,2Λ I]Compound 1, 2 or 3 binding is defined as total binding minus binding measured in the presence of 1-10 μM cold Compound 1, 2 op 3. The above asεay iε validated using the procedures outlined in paragraphs (a) - (f) of Example 8. Example 13 : Recombinant Receptor Binding Aεsay The following is one example of a rapid screening aεsay for useful compounds of this invention. In this aεεay, a cDNA or gene clone encoding the I 1O channel binding εite (receptor) from a suitable organism such as a human is obtained using standard procedures. Such receptors have been cloned and are known in the art. Diεtinct fragmentε of the clone are expreεεed in an appropriate expreεsion vector to produce the smallest polypeptide(s) obtainable from the receptor which retain the ability to bind Compound 1, 2 or 3. In thiε way, the polypeptide(ε) which includeε the novel Compound 1/ Compound 2/Compound 3 receptor for theεe compoundε can be identified. Such experiments can be facilitated by utilizing a stably transfected mammalian cell line (e.g. , HEK 293 cells) expresεing the I lo channel. Alternatively, the Compound 1/Compound 2/ Compound 3 receptor can be chemically reacted with chemically modified Compound 1, 2 or 3 in εuch a way that amino acid reεidueε of the Compound 1/Compound 2/Compound 3 peptide receptor which contact (or are adjacent to) the εelected compound are modified and thereby identifiable. The fragment(ε) of the Compound 1/Compound 2/Compound 3 receptor containing thoεe amino acidε which are determined to interact with Compound 1, 2 or 3 and are sufficient for binding to said molecules, can then be recombinantly expreεεed, aε described above, using a standard expresεion vector(ε) . The recombinant polypeptide(ε) having the deεired binding propertieε can be bound to a solid phase support using standard chemical procedures. This solid phase, or affinity matrix, may then be contacted with Compound 1, 2 or 3 to demonstrate that those compounds can bind to the column, and to identify conditions by which the compounds may be removed from the solid phase. This procedure may then be repeated using a large library of compounds to determine those compounds which are able to bind to the affinity matrix, and then can be releaεed in a manner similar to Compound 1, 2 or 3. However, alternative binding and release conditions may be utilized in order to obtain compounds capable of binding under conditions distinct from those used for Compound 1/ Compound 2/Compound 3 peptide binding (e.g. , conditionε which better mimic physiological conditions encountered, especially in pathological εtateε) . Thoεe compoundε which do bind can thuε be εelected from a very large collection of compoundε preεent in a liquid medium or extract. Once compoundε able to bind to the Compound 1/ Compound 2/Compound 3 binding polypeptide(ε) deεcribed above are identified, those compounds can then be readily tested in the various asεayε described above to determine whether they, or simple derivatives thereof, are useful compounds for therapeutic treatment of cardiac and neurological disorderε described above. In an alternate method, native Compound 1, 2 or 3 receptor can be bound to a column or other solid phase εupport. Thoεe compoundε which are not competed off by reagentε which bind other εites on the receptor can then be identified. Such compounds define novel binding sites on the receptor. Compounds which are competed off by other known compounds, thus bind to known siteε, or bind to novel εites which overlap known binding sites. Regardless, such compounds may be structurally distinct from known compounds and thuε may define novel chemical claεses of agonists or antagonist which may be useful aε therapeuticε. Formulation and Administration As demonstrated herein, useful compounds of this invention may be used to treat neurological diseaεes or disorders. While these compounds will typically be used in therapy for human patients, they may be used to treat similar or identical diseases in other vertebrates such as other primateε, farm animalε εuch aε swine, cattle and poultry, and sportε animals and pets such as horses, dogs and cats. In therapeutic and/or diagnoεtic applicationε, the compoundε of the invention can be formulated for a variety of modeε of adminiεtration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington 's Pharmaceutical Sciences , Mack Publishing Co. , Easton PA. For systemic administration, oral administration iε preferred. Alternatively, injection may be used, e.g. , intramuεcular, intravenouε, intraperitoneal, and εubcutaneous. For injection, the compounds of the invention are formulated in liquid solutionε, preferably in phyεiologically compatible bufferε εuch as Hank's solution or Ringer'ε solution. Alternatively, the com- pounds of the invention are formulated in one or more excipients (e.g.. propylene glycol) that are generally accepted as safe as defined by USP standardε. In addi¬ tion, the compoundε may be formulated in solid form and redissolved or εuspended immediately prior to use. Lyophilized forms are also included. Systemic administration can also be by transmucoεal or tranεdermal meanε, or the compoundε can be administered orally. For tranεmucoεal or tranεdermal adminiεtration, penetrantε appropriate to the barrier to be permeated are uεed in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile saltε and fuεidic acid derivativeε. In addition, detergents may be used to facilitate permeation. Transmucoεal administration may be through nasal sprays, for example, or using suppoεitories. For oral administration, the compounds are formulated into conventional oral adminiεtration forms such as capεules, tablets and tonics. For topical administration, the compounds of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art. The amountε of various compoundε of thiε invention which must be administered can be determined by standard procedure. Generally it is an amount between about 1 and 50 mg/kg of the animal to be treated. Other embodiments are within the following claims. "Sequence Listing" (1) GENERAL INFORMATION: (i) APPLICANT: Michael C. Sanguinetti Alan Mueller (ii) TITLE OF INVENTION: POTASSIUM CHANNEL BLOCKING COMPOUNDS AND THEIR USE (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Lyon & Lyon (B) STREET: 611 West Sixth Street (C) CITY: Los Angeles (D) STATE: California (E) COUNTRY: USA (F) ZIP: 90017 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: 3.5" Diεkette, 1.44 Mb storage (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: IBM MS-DOS (Version 5.0) (D) SOFTWARE: WordPerfect (Version 5.1) (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: Prior applications total, including application described below: 1 (A) APPLICATION NUMBER: 08/033,388 (B) FILING DATE: 03/18/93 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: WARBURG, RICHARD J. (B) REGISTRATION NUMBER: 32, 327 (C) REFERENCE/DOCKET NUMBER: 206/093 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (213) 489-1600 (B) TELEFAX: (213) 955-0440 (C) TELEX: 67-3510 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Asp Asp Cys Gly Lys Leu Phe Ser Gly Cyε Aεp Thr Aεn Ala 1 5 10 Aεp Cyε Cyε Glu Gly Tyr Val Cyε Arg Leu Trp Cyε Lyε Leu 15 20 25 Aεp Trp 30 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 (B) TYPE: amino acid (C) STRANDEDNESS: εingle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Glu Cyε Gly Thr Leu Phe Ser Gly Cys Ser Thr His Ala Asp 1 5 10 Cys Cys Glu Gly Phe lie Cys Lys Leu Trp Cyε Arg Tyr Glu 15 20 25 Arg Thr Trp 30 (3) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO:3: Asp Asp Cys Ala Gly Trp Met Glu Ser Cys Ser Ser Lys Pro 1 5 10 Cys Cys Ala Gly Arg Lys Cys Phe Ser Glu Trp Tyr Cys Lys 15 20 25 Leu Val Val Asp Gin Asn 30

Claims: Claimε 1. Specific potent tranεient outward potaεεiu channel inhibitorε. • 5 2. Method for screening for a transient outward potassium channel active agent, comprising the stepε of: contacting a said transient outward potassium channel with a known specific transient outward potasεium 10 channel inhibitor and a potential tranεient outward potaεsium channel active agent, and detecting inhibition of binding of said known εpecific tranεient outward potaεεium channel inhibitor by εaid potential tranεient outward potaεsium channel active 15 agent, wherein inhibition of binding is indicative of a useful transient outward potasεium channel active agent. 3. Method for treatment of a diεease or condition in which modulation of transient outward potaεεium channel 20 activity iε therapeutically uεeful, compriεing the εtep of: administering a therapeutically effective specific transient outward potassium channel inhibitor. 25 4. Method for treatment of a disease or condition in which modulation of transient outward potassium channel activity is therapeutically useful, comprising the step of: administering a therapeutically effective potasεium 30 channel inhibitor corresponding to an inhibitor present in a spider toxin. 5. The method of claim 4 wherein said channel iε a tranεient outward potaεεium channel. 6. Inhibitor obtainable from a εpider venom or unique fragment or analog of εaid polypeptide active to modulate a potassium channel. 7. Method for screening for a potassium channel active agent, comprising the stepε of: contacting a said potasεium channel with a known potaεεium channel inhibitor dε rived from εpider venom and a potential potaεsium channel active agent, and detecting inhibition of binding of said known inhibitor by said potential agent, wherein inhibition of binding iε indicative of a uεeful potassium channel active agent. 8. The method of claim 2 wherein said outward potassium channel inhibitor is selected from the group consiεting of Compound 1, Compound 2, and Compound 3. 9. The method of claim 3 wherein εaid tranεient outward channel inhibitor iε εelected from the group conεiεting of Compound 1, Compound 2 and Compound 3. 10. The method of claim 2 wherein said transient outward potaεεium channelε are from cardiac or neural tiεεue. 11. A compoεition conεiεting of Compound 3, or a pharmaceutically acceptable salt thereof. 12. A pharmaceutically acceptable compoεition compriεing a compound selected from the group consisting of Compound 1, Compound 2 and Compound 3. 13. An inhibitor of claim 1 selected from the group consisting of Compound 1, Compound 2 and Compound 3. 14. Method for the uεe of a potassium channel inhibitor isolated from spider venoms as an insecticidal agent, comprising the step of: applying to an insect or its environment an inhibitor present in spider venom.

Inventors: MUELLER ALAN L, SANGUINETTI MICHAEL C, MUELLER, ALAN, L., SANGUINETTI, MICHAEL, C.
Applicants: NPS PHARMA INC, NPS PHARMACEUTICALS, INC.
Application year: 1994
Main classification: A61K37
IPC codes: A01N63, A61K35, A61K38, A61K45, A61K49, A61P9, A61P43, C07K14, G01N33